Resistance to chemotherapeutic drugs and radiation is the most important factor in failures of conventional anticancer therapies to cure. A growing body of evidence suggests many human tumors contain a significant fraction of hypoxic cells that can directly affect therapeutic responsiveness. The detailed nature of these hypoxia- dependent resistance phenotypes remains unclear but involves a set of diverse proteins, called oxygen-regulated proteins (ORPs), whose expression is induced in response to transient and chronic hypoxia. This proposal is based on the hypothesis that the overexpression of a major isoform of metallothionein (MT-IIA) in hypoxic microenvironments of human tumors selects for malignant phenotypes. Proposed studies include experiments to precisely define the putative hypoxic responsive element(s) of the human MT-IIA 5'- regulatory region and to characterize the possible role of a metal-responsive element and a putative oxidant/ antioxidant response element in this regulation. The identities and properties of associated transcription factors will also be investigated. A second aim will extend ongoing studies of the cytoprotective role of transiently overexpressed MTs induced by hypoxia to subsequent exposures to reoxygenation, antineoplastic agents, or radiation exposure. This cytoprotective effect will be investigated by clonogenic and apoptosis assays, analysis of MI subcellular distributions, and monitoring of MT and apoptosis-associated protein expression patterns in a series of experiments using transgenic MT wild-type and knockout mouse cells and antisense-treated human squamous carcinoma and hepatoma cells. A detailed understanding of the genetic regulation and mechanisms of MT involvement in drug and radiation resistance in stressed regions of solid tumors will result in novel prognostic and therapeutic strategies. For example, these studies will provide a foundation for creating gene therapy approaches to cancer in which vector constructs containing the hypoxic/redox response elements that activate genes can be used for sensitizing tumor cells to other therapeutic agents.